Leveraging Malaysia’s Biodiversity towards Value Creations ... · PDF fileStructure...

Habibah A Wahab, PhD

Malaysian Institute of Pharmaceuticals and Nutraceuticals,

Ministry of Science, Technology and Innovation

SAINS@USM, 11900, Penang

[email protected]

Pharmaceutical Design and Simulation Laboratory,

School of Pharmaceutical Sciences, USM

[email protected]

Shaharum Shamsuddin D. Phil (Oxon.)

Advance Molecular and Cellular Biology Laboratory

Deputy Dean

School of Health Sciences, USM

[email protected]

Leveraging Malaysia’s

Biodiversity towards Value

Creations using Bioinformatics

mailto:[email protected]

mailto:[email protected]

World’s Megadiversity

Taxonomic Group Species Endemic Species Percent Endemism

Plants 25,000 15,000 60.0

Mammals 380 172 45.3

Birds 769 142 18.5

Reptiles 452 243 53.8

Amphibians 244 196 80.3

Freshwater Fishes 950 350 36.8

Biodiversity hotspot: Sundaland

http://www.biodiversityhotspots.org/xp/hotspots/sundaland/Pages/biodiversity.aspx

Country Mammals Birds Plants

Malaysia 337 746 15500

Malaysia Biodiversity and National Biotechnology Policy

► Innovation to create wealth by utilising and advancing

biotechnology for socio-economic benefits of the nation in

accordance with established social and ethical norms

Mission

Statement

► Position biotechnology as the new economic engine to enhance

prosperity and wellness of the nation by 2020

► Through Healthcare, Industrial and Agriculture

► Enhance R&D and Tech Acquisition, Human Capital Development,

Legal and Financial Structure

Vision

To optimise economic benefits from

sustainable utilisation of the components

of biological diversity;

• Identification and

Development of Bioactive

Compounds

• Bioprocessing

• Pre-formulation for Product

Development

• Screening of Bioactive

Compounds

• Advanced Drug Delivery

Systems

IPHARM

Wealth from plant biodiversity

• Food (through agriculture and harvest of natural population)

– Crops, livestock, forestry and fish

– Food products: fruits, vegetables, nuts

– Food Additives: colourings, flavourings, preservatives

• Medicine

– ~ 119 pure chemicals are extracted from < 90 species of higher plants and used as medicines

throughout the world, for example caffeine, quinine, vincristine, vinblastin.

• Industry

– Fibers for clothing; wood for shelter and warmth

– Source of energy (biomass);

– Industrial products: oils, lubricants, perfumes, fragrances, dyes, paper, waxes, rubber,

latexes, resins, poisons

– All these can be derived from various plant species.

• Tourism & Recreation

– Biodiversity is a source of economical wealth for many regions of the world

– Nature reserves, parks and forests

– Ecotourism, National park, wetland resort

Natural Products Discovery System (NADI) & Value creations

• Natural products

• Value creations

– Pharmaceuticals

– Nutraceuticals

– Cosmeceuticals

– Specialty chemicals

Natural product based pharmaceutical discovery

• The global pharmaceutical markets worth $770 billion in 2008 and expected to grow 4 to 6% in 2010 exceeding $825 billion following stable demand in 2009 (IMS report, June 2009).

• Natural products are established and long source of drug. 61% of the 877 small molecule

new chemical entities introduced as drugs worldwide during 1981-2006 can be traced to or

were inspired by natural products.

David J. Newman; Gordon M. Cragg; J. Nat. Prod. 2007, 70, 461-477.

Natural Products: Source of Drugs (& medications)

Natural Product Drug Discovery: Opportunity

• ~250,000 flowering plant known , ~ 125K found in the tropical forests.

• Only ~1% of tropical species have been studied for their pharmaceutical potential.

• 2002-2006, 26 are plant derived.

BioMalaysia2009, 18th Nov. 2009, KLCC, Kuala Luampur

Natural Product Discovery: Opportunity

• Malaysia has > 15000 flowering species, 2000 with medicinal values

• Many compounds (vincristine, vinblastine, reserpine) in Malaysian natural products are

current drugs in the market though not discovered in Malaysia.

• Herbal industry in Malaysia, RM8 billion/year – growing at 10% per year.

39%

6%

27% 5%

23%

61%

Natural Products

NatProd Deriv ativ es

Sy nthetic compounds w ith NatProd deriv ed pharmacophore

NatProd mimic

Antibacterials 78%

Anticancer 74%

Used to treat 87%

(48/55)

of human diseases

David J. Newman* and

Gordon M. Cragg J. Nat.

Prod., 70 (3), 461 -477,

2007”

http://pubs.acs.org/cgi-bin/sample.cgi/jnprdf/2007/70/i03/html/np068054v.html

Challenges • Approaches to natural discovery:

– random selection followed by chemical screening,

– random selection followed by one or more biological assays, follow-up of biological

activity reports,

– follow-up of ethnomedical (traditional medicine) use of plants

• Challenges:

– Tedious isolation and characterization of natural products and heavy reliance on good

screening and bioassays

– The discovery of compounds that are cytotoxic or have other unsuitable properties.

– Reinventing the wheels

• Problems:

– not enough availability. can be overcome by semi-synthesis/synthesis or using tissue-

culture techniques

– Isolated bioactive compounds are known compounds

NADI: Bioinformatic approach to complementing Natural Product Research

• NADI applications:

– One stop centre for information

– Plant selections • Ethnopharmological basis & Follow-on research

• Systematic evaluation

• Modelling?

– Assay selections • Ethnomedical basis

• Random/systematic screening

• Virtual Screening?


NADI:

Bioinformatic databases: small

molecules, receptor databases,

plant monographs

Bioinformatic tools: Blast, Protein

Structure Prediction, Molecular

Docking etc.

NADI: Information Centre for Malaysian Herbs


Ethnomedical/follow-on basis

Keyword Search:

Symptom Keyword Search: Plant’s name

Plant’s name

Plant’s part used

Introduction about the plant


Traditional Uses of the plant

Scientific Studies on Bioactivities

References


Live search on the PubMed on

the related references

From the monographs:

Tongkat Ali has been used:

•Aphrodisiac

•Fever

•Wound healing

•Headache

•And many more

•Scientific studies:

•Increase testosterone

•Anti-malaria

• Antileucaemiac.

•Antitumor

•Antipyretic.

• Antiulcer


Systematic Evaluation

Who’s doing what?


• What has been done? • Do we need to reinvent the wheel?

No of compounds in a plant

0

20

40

60

80

100

120

140

Aca

lyph

a indica

Alpinia raffle

sia

And

rograph

is pan

iculata

Artoc

arpu

s he

tero

phyllus

Bau

hinia malab

arica

Blumea

luce

ra

Cae

salpinia pulch

errim

a

Cas

sia alata

Cen

tella

asiatica

Citrullus vu

lgar

is

Citrus

aur

antiu

m

Citrus

clemen

tina

Citrus

limon

Claus

ena exc

avat

e

Cur

cum

a long

a

Cur

cum

a ze

doa

ria

Cyp

erus ro

tund

us

Eug

enia cum

ini

Eug

enia m

iche

lii

Garcinia atro

virid

is

Har

risonia pe

rfora

te

Jasm

inum

sam

bac

Miche

lia A

lba

Mitragy

na spe

cios

a

Mor

inda

citr

ofolia

Myristic

a fra

grans

Orth

osiph

on stamineu

s

Pan

danu

s am

aryllifolius

Piper

betle

Piper

retro

frac

tum

Pre

mna

tomen

tosa

Ros

a da

mas

cena

Sm

ilax myo

sotiflora

Tab

erna

emontan

a diva

ricata

Trig

onella foe

num

Zingibe

r ca

ssum

unar

Zingibe

r ze

rum

bet

Plant

No

of

co

mp

ou

nd

s

Series1


Rational Selection – Assays

• When a novel compound identified, how do I know it has potential as a drug?

• NADI provides link to USM’s Reverse Docking for prediction of potential receptor(s) to target….

Molecular

Dynamics

Simulation

Molecular

Docking

Virtual Hits

Identified

In vitro

assay

Hit Identified

Literature

review Pharmacop

hore -

Modelling

Lead Identified

Virtual Lead

Identified

Further tests

NADI Drug Design Workflow

Validated target

Identified

*

*

Ensemble Based Docking

AutoDock

Set of docked

complexes, BEs

Post-processing

ranking schemes

Ligand PDBs

Ligands

ZINC

available

NADI NCI

N/A

Org.

synth

Receptor xtal struct

Explicit

MD

Snapshot

10 ps

Receptor ensemble

R. Amaro, JACS, 2008

NADI example: TB

• Caused by Mycobacterium tuberculosis

• One third of the world population is infected

• WHO: 8 million new cases each year

: 30 million people will die of tb this decade

• As much as 30% INH resistant strain TB

• Multi-drug resistant strain in HIV/AIDS patients

Drug Target: InhA (PDB ID: 1ZID)

I47 V78 I95

S94A MT InhA (PDB ID: 1ENZ) I16 I21

Wahab, JCIM, 2009

in-silico screening using NADI

~400 plants,

~4000 compounds


INADH-The

ultimate

inhibitor

Virtual Hits Identified

Plants Identified

Table 1: The predicted activities of chemical compounds

reportedly to be isolated from Malaysian plants

Compounds Plant source Predicted Inhibition Constant (Ki)

D Pegaga 5.78E-17

B Akar susun kelapa 4.73E-16

C Sireh

4.13E-16

E Kunyit 1.5E-16

A Bunga tahi ayam

1.05E-16

Isoniazid (INADH) Synthetic molecule 3.795E-13

A B

C

D

E

No.

Plants Plant parts

Extracts MIC

(µg/ml)

1 Lantana camara (Bunga tahi ayam)

leaves Methanol 25

2 Centella asiatica (Pegaga)

whole plant

Methanol 25

3 Psidum guajava (Jambu batu)

leaves Methanol 200

fruits Methanol 50

4 Tabernaemontana coronaria (Jasmin)

leaves Methanol 50

5 Phyllanthus niruri (Dukung anak)

whole plant

Methanol 200

6 Murraya paniculata (Kemuning)

leaves Methanol 50

7

Piper betle (sireh)

whole plant

Ethanol 100

Chloroform 50

Petroleum ether

50

Ethanol:water 50

8 Isoniazid

0.31

CONTROL

Minimum inhibitory concentration (MIC) determinations

NADI example 2 : Avian Influenza

•Major outbreak began 2003.

Affected countries:

•The Republic of Korea, Viet Nam,

Japan, Thailand, Cambodia, The Lao

People’s Democratic Republic,

Indonesia, China, and Malaysia.

•2005, the virus spread to the

Russian Federation, Kazakhstan,

Mongolia, Turkey, Romania,

Croatia, Ukraine and the

Netherland.

•Japan, the Republic of Korea,

and Malaysia have controlled

their outbreaks and are now

considered free of the disease.

Elsewhere in Asia, the virus has

become endemic in several of

the initially affected countries.

•Source:

WHO

viral replication and drug targets

• Influenza virus membranes contain two glycoproteins: haemagglutinin and neuraminidase.

• 2 groups of neuraminidase subtypes: – group-1 contains the subtypes N1, N4, N5 and

N8 (Avian Flu is of N1 subtype). – group-2 contains the subtypes N2, N3, N6, N7

and N9.

• The enzyme facilitates the spread of virus during an infection, thus becomes an attractive target for antiviral drugs. E.g. of drugs inhibit its activity are oseltamivir and zanamivir.

• These inhibitors were originally developed using crystal structures of neuraminidase subtypes N9 and N2 and another neuraminidase from the type B genus of influenza viruses.

• Other drug targets include M2, H2 and RNA polymerase.

Total: 208 plants

3500 cpds

in-silico screening using NADI


Docking of Oseltamivir onto H5N1 Neuraminidase

• RMSD: 1.3 Å

• Predicted Ki = 0.13 nM (exp Ki ranging from 0.073 – 0.37 nM, AAC, Aug. 2006)

Compound name Final Docked energy,

kcal/mol Inhibition constant, Ki

MSC 986 -11.27 5.35E-09

MSC 1026 -12.54 3.45E-09

MSC 1685 -12.71 6.29E-09

MSC 1880 -15.55 8.58E-10

MSC 1941 -12.22 9.42E-10

Top 5 compounds screened from MD trajectories of neuraminidase

MSC986 MSC1026

MSC 1685

MSC 1880

MSC 1941

Preliminary results of enzyme Inhibition Studies % Inhibitory of Neuraminidase by

methanol extract

0

20

40

60

80

100

120

0 5 10 15

Sample Concentration

% i

nh

ibit

ory

Sample Concentration (µg/ml)

% inhibitory i % inhibitory ii Average

12.00 95.50 95.80 95.65

3.00 83.20 84.90 84.55

1.50 59.80 77.10 68.45

0.75 67.80 72.10 69.95

0.05 47.60 49.70 48.65

0

20

40

60

80

100

120

50 100 500 1000 2000 3000 5000

Concentration (ug/mL)

% I

nh

ibit

ion

THF-pet ether extract

Oxalomalic acid

Oseltamivir

Plaque assay experiment : Testing the compound againts live H1N1 virus

NADI, Herbals, Cosmetics and Specialty Chemicals

Natural Product Discovery: Way Forward

• NADI database and bioinformatics approach important and have values to:

– the rational selection of plants for further studies – survey new chemical structures found in various classes of natural compounds – predict compounds with new pharmacological activity (compounds only have

value if they act differently or on new targets relative to known structures) – utilise natural products derived structures as guiding principle for new

chemical library for synthesis. • Analogues of natural products can be more potent than the parent compounds, or

possess superior drug-like properties. • New biological activities not even seen with the parent molecule. • Intensified search for new natural product derived molecules.

• Collaboration efforts from different disciplines – rapid and cost-effective discovery

• Safeguard our natural treasure (wealth, security)

Acknowledgement

NADI Developers: NAPIMM:

Yam Wai Keat, Nurul Bahiyyah, Suhaini Ahmad

NADI-RA

Nur Hanani Che Mat

NADI-HERBS

Nornisah Mohamed

NADI-CHEM

Suhaini Ahmad, Ezatul E Kamarulzaman, Ros Fatihah Halim, Nur Faezah Abdullah

NADI-VISAGE

• Suhaini Ahmad,

• Junya Seo (PhD,

Osaka U)

• Mitsuru Jikeya (MSc,

Osaka U)

• Masafumi Tominaga

(MSc, Osaka U)

Post Doctoral Fellow Dr. Hassan Abdullah

Dr Muhamad TM Al Dajani

Postgraduates: Mohd Razip Asaruddin

(PhD)

Suhaini Ahmad (MSc)

Nurul Izzah (MSc)

Noor Hamdah Musa (MSc)

Siti Marina Maidin (MSc)

Sharizan Abdullah (MSc)

Fauziah Hanim (MSc)

Fauziahanim Zakaria (MSc)

Internship Students:

Vicky Yang (BSc, UCSD)

Cindy Trans (BSc, UCSD)

Ranmali Varahenage

(BSc, UC San Diego)

BIRD FLU RESEARCH GROUP

Academic PI

•Dr. Aisyah Saad Abdul Rahim

•Dr. Hasnah Osman

•Dr. Shafida Abdul Hamid

•Dr. Nornisah Mohamed

•Dr. Razip Samian

•Dr. Normi Yusuf

•Dr. Rashidah Abdul Rahim

•Dr. Shaharum Shamsuddin

•Dr. Sharifah Syed Hassan

Funding

USM Research University Grant

TB Drug Discovery

•Dr. Shaida Fariza Sulaiman

•Suriyati Muhamad

•Dr. Pazilah Ibrahim

•Dr. Choong Yee Siew

Funding

MOSTI-EScience

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